Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy.

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

Secretion of functional α1-antitrypsin is cell type dependent: Implications for intramuscular delivery for gene therapy.

Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.

Delivery of Adeno-Associated Virus Vectors to the Central Nervous System for Correction of Single Gene Disorders.

Genetic disorders of the central nervous system (CNS) comprise a significant portion of disability in both children and adults. Several preclinical animal models have shown effective adeno-associated virus (AAV) mediated gene transfer for either treatment or prevention of autosomal recessive genetic disorders. Owing to the intricacy of the human CNS and the blood-brain barrier, it is difficult to deliver genes, particularly since the expression of any given gene may be required in a particular CNS structure or cell type at a specific time during development. In this review, we analyzed delivery methods for AAV-mediated gene therapy in past and current clinical trials. The delivery routes analyzed were direct intraparenchymal (IP), intracerebroventricular (ICV), intra-cisterna magna (CM), lumbar intrathecal (IT), and intravenous (IV). The results demonstrated that the dose used in these routes varies dramatically. The average total doses used were calculated and were 1.03 × 1013 for IP, 5.00 × 1013 for ICV, 1.26 × 1014 for CM, and 3.14 × 1014 for IT delivery. The dose for IV delivery varies by patient weight and is 1.13 × 1015 IV for a 10 kg infant. Ultimately, the choice of intervention must weigh the risk of an invasive surgical procedure to the toxicity and immune response associated with a high dose vector.

Gene-based therapeutics for rare genetic neurodevelopmental psychiatric disorders.

We are in an emerging era of gene-based therapeutics with significant promise for rare genetic disorders. The potential is particularly significant for genetic central nervous system disorders that have begun to achieve Food and Drug Administration approval for select patient populations. This review summarizes the discussions and presentations of the National Institute of Mental Health-sponsored workshop "Gene-Based Therapeutics for Rare Genetic Neurodevelopmental Psychiatric Disorders," which was held in January 2021. Here, we distill the points raised regarding various precision medicine approaches related to neurodevelopmental and psychiatric disorders that may be amenable to gene-based therapies.

Muscle-Directed Delivery of an AAV1 Vector Leads to Capsid-Specific T Cell Exhaustion in Nonhuman Primates and Humans.

With the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approvals for Zolgensma, Luxturna, and Glybera, recombinant adeno-associated viruses (rAAVs) are considered efficient tools for gene transfer. However, studies in animals and humans demonstrate that intramuscular (IM) AAV delivery can trigger immune responses to AAV capsids and/or transgenes. IM delivery of rAAV1 in humans has also been described to induce tolerance to rAAV characterized by the presence of capsid-specific regulatory T cells (Tregs) in periphery. To understand mechanisms responsible for tolerance and parameters involved, we tested 3 muscle-directed administration routes in rhesus monkeys: IM delivery, venous limb perfusion, and the intra-arterial push and dwell method. These 3 methods were well tolerated and led to transgene expression. Interestingly, gene transfer in muscle led to Tregs and exhausted T cell infiltrates in situ at both day 21 and day 60 post-injection. In human samples, an in-depth analysis of the functionality of these cells demonstrates that capsid-specific exhausted T cells are detected after at least 5 years post-vector delivery and that the exhaustion can be reversed by blocking the checkpoint pathway. Overall, our study shows that persisting transgene expression after gene transfer in muscle is mediated by Tregs and exhausted T cells.

A Safe and Reliable Technique for CNS Delivery of AAV Vectors in the Cisterna Magna.

Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.

Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema.

Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is α-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a-e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research.

Class I-restricted T-cell responses to a polymorphic peptide in a gene therapy clinical trial for α-1-antitrypsin deficiency.

Adeno-associated virus (AAV)-mediated gene therapy is currently being pursued as a treatment for the monogenic disorder α-1-antitrypsin (AAT) deficiency. Results from phase I and II studies have shown relatively stable and dose-dependent increases in transgene-derived wild-type AAT after local intramuscular vector administration. In this report we describe the appearance of transgene-specific T-cell responses in two subjects that were part of the phase II trial. The patient with the more robust T-cell response, which was associated with a reduction in transgene expression, was characterized more thoroughly in this study. We learned that the AAT-specific T cells in this patient were cytolytic in phenotype, mapped to a peptide in the endogenous mutant AAT protein that contained a common polymorphism not incorporated into the transgene, and were restricted by a rare HLA class I C alleles present only in this patient. These human studies illustrate the genetic influence of the endogenous gene and HLA haplotype on the outcome of gene therapy.

The rapidly evolving state of gene therapy.

Gene therapy is emerging as a viable option for clinical therapy of monogenic disorders and other genetically defined diseases, with approved gene therapies available in Europe and newly approved gene therapies in the United States. In the past 10 years, gene therapy has moved from a distant possibility, even in the minds of much of the scientific community, to being widely realized as a valuable therapeutic tool with wide-ranging potential. The U.S. Food and Drug Administration has recently approved Luxturna (Spark Therapeutics Inc, Philadelphia, PA, USA), a recombinant adeno-associated virus (rAAV) 2 gene therapy for one type of Leber congenital amaurosis 2 ( 1 , 2 ). The European Medicines Agency (EMA) has approved 3 recombinant viral vector products: Glybera (UniQure, Amsterdam, The Netherlands), an rAAV vector for lipoprotein lipase deficiency; Strimvelis (Glaxo Smith-Kline, Brentford, United Kingdom), an ex vivo gammaretrovirus-based therapy for patients with adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID); and Kymriah (Novartis, Basel, Switzerland), an ex vivo lentivirus-based therapy to engineer autologous chimeric antigen-receptor T (CAR-T) cells targeting CD19-positive cells in acute lymphoblastic leukemia. These examples will be followed by the clinical approval of other gene therapy products as this field matures. In this review we provide an overview of the state of gene therapy by discussing where the field stands with respect to the different gene therapy vector platforms and the types of therapies that are available.-Gruntman, A. M., Flotte, T. R. The rapidly evolving state of gene therapy.

AAV9 gene replacement therapy for respiratory insufficiency in very-long chain acyl-CoA dehydrogenase deficiency.

Very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an autosomal recessive disorder of fatty acid oxidation. Fatty acids are a major source of energy during catabolic stress, so the absence of VLCAD can result in a metabolic crises and respiratory insufficiency. The etiology of this respiratory insufficiency is unclear. Thus, our aims were: (1) to characterize respiratory pathophysiology in VLCADD mice (VLCAD-/- ), and (2) to determine if AAV9-mediated gene therapy improves respiratory function. For the first aim, VLCAD-/- and wild-type (WT) mice underwent an exercise/fast "stress protocol" and awake spontaneous breathing was evaluated using whole-body plethysmography (WBP) both at baseline and during a hypercapnic respiratory challenge (FiO2 : 0.21; FiCO2 : 0.07; nitrogen balance). During hypercapnia, VLCAD -/- mice had a significantly lower frequency, tidal volume, minute ventilation, and peak inspiratory and expiratory flow, all of which indicate respiratory insufficiency. Histologically, the cardiac and respiratory muscles of stressed VLCAD -/- animals had an accumulation of intramyocellular lipids. For the second aim, a single systemic injection of AAV9-VLCAD gene therapy improved this respiratory pathology by normalizing breathing frequency and enhancing peak inspiratory flow. In addition, following gene therapy, there was a moderate reduction of lipid accumulation in the respiratory muscles. Furthermore, VLCAD protein expression was robust in cardiac and respiratory muscle. This was confirmed by immuno-staining with anti-human VLCAD antibody. In summary, stress with exercise and fasting induces respiratory insufficiency in VLCAD-/- mice and a single injection with AAV9-VLCAD gene therapy ameliorates breathing.

Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of α-1 Antitrypsin Deficiency.

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated.

Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood-Onset Retinal Dystrophy.

PURPOSE: To provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations. DESIGN: Nonrandomized, multicenter clinical trial. PARTICIPANTS: Eight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood-onset retinal degeneration (SECORD). METHODS: Patients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment. MAIN OUTCOME MEASURES: The primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire. RESULTS: All patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area. CONCLUSIONS: Treatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns.